There is an increasing need for simple, rapid and easy-to-use sensors to aid clinical diagnosis and for environmental analysis.
The very specific and tight binding between antibodies and antigens has traditionally been measured in clinical laboratories by radioimmunoassay, solid phase enzymeimmunoassay and fluoroimmunoassay. Quantification of the antibody-antigen complex relies on a marker molecule, such as a radioisotope, an enzyme or a fluorescent probe. In most cases, the result is not obtained until several incubations, washing and separation steps have been performed.
Presently, immunosensors that can monitor antigen concentrations in real-time with the aid of an immobilized antibody and that do not require the use of labeled species are being developed. The lack of control over the orientation of the antibodies, however, limits the proportion of available binding sites when using conventional immobilization methods [Attili, B.; Suleiman, A.; Microchemical Journal 54 (1996) 174]. Site-specific immobilization, on the other hand, leads to higher activity. Options for controlled immobilization of antibodies include specific binding of the Fc region of the antibody to a protein A or protein G, covalent attachment through the free sulfhydryl group on the antibody Fab′-fragments onto supported layers and biotinylated antibodies coupled onto a surface by biotin/(strept)avidin chemistry [Morgan, H.; Taylor, D. M.; Biosensors & Bioelectronics 7 (1992) 405; Vikholm, I.; Albers, W. M.; Langmuir 14 (1998) 3865; Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E.; Langmuir (9) (1993) 136; Schönhoff, M.; Lösche, M.; Meyer, M.; Wilhelm, C.; Progr. Colloid Polym. Sci. (1992) 243; Krull, U. J.; Brown, R. S.; Vandenberg, E. T.; Heckl, W. M.; J., Electr. Microscopy Technique 18 (1991) 212; Heyn, S. P.; Egger, M.; Gaub, H. E.; J. Phys. Chem. 94 (1990) 5073].
It has, however, recently been shown that Fab′-fragments can be adsorbed directly onto gold with high epitope density [O'Brien, J.; Jones, V.; Porter, M.; Anal. Chem. 72 (2000) 703]. The high surface energy of metal surfaces normally leads to denaturation of biomolecules with a decrease in activity. The mechanism of adsorption has been studied extensively and involves hydrophobic interaction between the surface of the material and hydrophobic patches on the surface of the biomolecule. After the initial adsorption, the protein may denaturate and unfold to expose more hydrophobic groups and thus lose its activity [Andrade, J. D., Hlady, V., Adv. Polym. Sci. 79 (1986) 1-63].
Patent documents describing the use of various linker molecules which form a monolayer on a surface for attachment of biomolecules to make a biosensor where the detection can be performed by surface plasmon resonance, include U.S. Pat. No. 5,242,828 and European Patents nos. 442922 and 485874.
Receptor molecules, which could be attached onto the transducer interface in an oriented and reproducible manner with minimal unfolding, would enable optimal preservation of the activity and specificity and could be used in sensor applications requiring high sensitivity. A site-directed immobilization of the receptor molecules would ensure maximal binding efficiency.
Another problem that has to be overcome in biosensor applications is non-specific binding. Biorepellent surfaces have become a subject of great interest in fields such as biochemistry, biology, biotechnology, and health care, because of the need to control the interaction of biomolecules and cells with various surfaces. Protein-repellent surfaces are crucial to blood-contacting implant devices, membranes for separation processes, chromatographic supports, contact lenses, and blood and protein storage devices.
Biomolecule repellent surfaces can be prepared by immobilizing nonionic, hydrophilic polymers, such as poly(acrylamide), poly(N,N-dimethylacrylamine), poly(vinylalcohol), ethylene-vinylalcohol copolymer, poly(hydroxyethyl methacrylate), poly(ethyleneoxide) and poly(ethyleneglycol) [Köberle, P.; Laschewsky, A.; van den Boogaard, D.; Polymer 33 (1992) 4029; Saito, N.; Sugawara, T.; Matsuda, T.; Macromolecules 29 (1996) 313]. The resistance to adsorption of proteins is based on the entropic and hydrodynamic repulsion of the flexible molecular chains of the biorepellent surface and the biomolecules of a solution being higher than the combined forces of attraction. Polymers have been adsorbed by physical coating, chemical coupling or graft copolymerisation.
Self-assembled alkanethiols that present oligo(ethyleneglycol) have been mixed with molecules possessing suitable linker groups, onto which biomolecules can be attached for biosensor application [E. Ostuni, L. Yan, G. Whitesides, Colloids and Surfaces B: Biointerfaces 15 (1999) 3-30].